Nucleolar dominance describes the transcription of one parental set of rRNA genes, and the silencing of the other parental set, in a plant or animal inter-species hybrid. Recent evidence shows that an epigenetic switch involving concerted changes in DNA methylation, histone methylation and histone acetylation controls the activation/silencing decision. Using Arabidopsis suecica, the hybrid of A. thaliana and A. arenosa, the shortterm goal is to test the hypothesis that rRNA gene promoter DNA methylation, histone deacetylation and histone H3 lysine 9 (H3K9) methylation are each upstream of one another in a self-reinforcing cycle that maintains silencing. Two histone deacetylases (one localized in the nucleolus), one DNA methyltransferase and several methylcytosine binding proteins required for nucleolar dominance are already in hand. Additional chromatin modifying proteins required for silencing will be identified using RNAi-mediated gene knockdowns. Biochemical specificities of these activities will be determined and the hypothesis that they interact at silenced genes will be tested using chromatin immunoprecipitation, protein-protein interaction assays and cytological localization. Involvement of silencing activities in both establishment and maintenance will be tested during development and in newly formed hybrids. The hypothesis that methylation of specific promoter cytosines is key to silencing will also be tested. These efforts will help achieve the long-term goal of understanding how nucleolar dominance is established and enforced. DNA methylation and chromatin modifications are required for proper development. In knockout mice, DNA methyltransferase and histone H3K9 methylase activities are essential for viability. In humans, mutations in a DNA methyltransferase causes ICF syndrome and mutations in a histone deacetylase associated methylcytosine binding protein causes Rett syndrome. DNA methylation is also frequently elevated in tumors and can silence tumor suppressor genes. By dissecting mechanisms of chromatin-mediated silencing in nucleolar dominance, we hope to contribute to the understanding, and ultimately the treatment, of disorders involving aberrant gene expression.